Program Summary Dengue is the most common arthropod borne viral disease of humans. Vaccines are urgently needed to prevent dengue yet vaccine development is complicated by the presence of four dengue virus (DENV) serotypes and the possibility of immune enhanced dengue disease. The leading vaccine candidates contain 4 live attenuated viruses to cover the 4 serotypes. The most advanced vaccine candidate, a chimeric Yellow Fever-Dengue tetravalent live virus vaccine (CYD-TDV) developed by Sanofi Pasteur, was recently evaluated in human efficacy studies conducted in Asia and Latin America. Overall, CYD-TDV was efficacious at reducing the burden of dengue disease in the vaccinated populations. However, the vaccine had unexpectedly low efficacy against DENV serotype 2 (DENV2) and in dengue nave subjects compared to dengue exposed subjects who were vaccinated. The lower efficacy in these groups was unexpected because the vaccine induced neutralizing antibodies (Abs) in these subjects. The central hypothesis of this proposal is that the quality (Ab epitope specificity) rather than total quantity of cell-culture neutralizing Abs is a better predictor of DENV vaccine performance in human populations. Moreover, as the DENV complex has 4 serotypes and vaccines will be used in populations with a mix of nave and partially immune individuals, immune assays based on a single epitope are unlikely to predict efficacy against the 4 serotypes. This project is grounded on studies in our laboratories to understand protective and pathogenic Ab responses in people exposed to natural DENV infections. We have discovered new quaternary structure Ab epitopes linked to protection and developed reagents (human monoclonal Abs, recombinant DENVs) and assays that precisely measure Ab epitope-specific responses in human sera. In collaboration with Sanofi Pasture, we will use samples from the CYD-TDV efficacy trials, including several hundred samples from people who experienced vaccine breakthrough infections, to identify Ab epitope based correlates of protective immunity. Currently we are the only group capable of doing this study because no other dengue vaccine has been tested in efficacy studies. As DENV vaccines have 4 attenuated replicating viruses, it has been difficult to obtain balanced replication and immunity to all 4 serotypes. Currently there are no reliable models to optimize the formulation of multi-component live dengue vaccine formulations. Investigators at Sanofi Pasteur's VaxDesign campus have developed a fully automated human Peripheral Tissue Equivalent (PTE) biomimetic model for preclinical evaluation of vaccines. As DENVs initially replicate in peripheral tissues after mosquito transmission or vaccination, we will test if the PTE biomimetic model has utility for predicting and optimizing the replication of single and multicomponent live-attenuated dengue vaccines. Within the time frame of this study, we will establish novel standardized assays for supporting global efforts to develop dengue vaccines.